Immunoadsorber for use in sepsis therapy

ABSTRACT

The invention relates to immunoadsorbers for use in sepsis therapy, in particular for removal of complement factors and lipopolysaccharides (LPS) and, if need be, further sepsis mediators such as TNF and interleukins from body fluids, methods for their production and their use.

The invention in question relates to an immunoadsorber for use in sepsistherapy, in particular for removing complement factors andlipopolysaccharides (LPS) as well as, if need be, TNF and interleukinsfrom body fluids and methods for their production and their use.

Every year, about 3.5 million patients suffer from sepsis in the USA,Japan and the EU. With a total number of inhabitants of 785 million, theincidence for these countries is less than 0.5%. But when hospitalisedpatients are examined with regard to the frequency of suffering,2.0±0.16 cases of sepsis are found per 100 hospital admissions. Theenormous health political and individual importance can also be seenfrom the observation that about 25% of these patients also suffer thesyndrome of a septic shock, characterised by the lethality rate of >45%,even with most intensive medicinal care by highly qualified specialistsin institutions with modern equipment (intensive care units).

The risk of suffering a septic shock is very high especially withpoly-traumatised patients (traffic accidents, burns, seriousoperations). Alongside infection from the outside, breaking through theintestinal barrier for gram-negative bacteria normally occurring in theintestines as a result of a partial loss of function of the immunesystem of these patients and thus an infection “from the inside” can bedetected.

In more than 50% of the cases, gram-negative bacterial or theircell-wall components, endotoxins (lipopolysaccharides, LPS), cause theseptic shock. The LPS released by bacterial binds to a serum protein(LBP) and is then absorbed by the LPS receptors of themonocytes/macrophages (CD14). The CD14+ cells activated in this wayproduce cytokines (TNFα, Interleukin-1′ (IL-1), IL-6, IL-8), which havetheir effect via cytokine receptors of the target cells.

Parallel to the stimulation of the monocytes and macrophages, thecomplement system is activated. It is an integrated part of theimmunological defence of mammals for direct and unspecific combating ofbacterial micro-organisms and foreign particles. Of the complementproteins occurring in the blood serum, primarily proenzymes activated byproteolytic fission, the C3 protein with a serum concentration of about1 g/l plays a central role. After contact of the micro-organisms withthe C3, the complement protein C3a is split off and, on the one hand,the formation of C5 convertase is initiated by the resultant C3b(alternative way of complement activation) and, on the other hand, thereaction is amplified by the C3B converting to C3 convertase due todepositing of serum factors. The complement protein C5 also occurring inserum is now proteolytically fissured by the C5 convertase, which isprovided in larger amounts, also forming C5a. Further complementproteins (C6-C9) deposit on the resulting C5b until finally thepolymeric hydrophobic membrane attack complex (MAC) is formed, settlingin the bacteria membrane (opsonidisation) and forming pores, which leadto phagocytosis and thus to the elimination of the micro-organisms (andthe bound MAC). The complement factors C3a and C5A (anaphylatoxins)released in the process of the complement activation result instimulation of the phagocytising cells to the location of the bacterialattack by increasing the vascular permeability and the release ofchemotoxins induced thereby. The reduction of the number of bacteriaresults in a reduction of the activation of the complement system. Thisdirect and unspecific reaction is closely connected with the otherimmunological defence systems insofar as the synthesis and release ofthe cytokines essential for cellular defence is regulated, for exampleby complement factors. In order to bring about the inflammatory effect,C3a and C5a are bound to specific cell-based receptors, which for theirpart are expressed in different strengths as a function of the immunereactivity. In order to keep the immune defence permanently ready foractivity, activated complement factors are detectable not only after anattack with micro-organisms, but also an integrated part of the serum ofstandard persons with a concentration of 1-10 ng/ml.

The plasma levels of the anaphylatoxins can be increased by a factor ofmore than one thousand, particularly in a developed sepsis, acutepulmonary failure and in moribund patients.

Almost exclusively on the basis of in vitro examinations, there existvarious, mainly unspecifically effective variations of solutions inorder to eliminate the effects of various complement factors, whichhowever can hardly be tested under in vivo conditions on account of theside effects to be expected (e.g. WO-A-98/34959).

In ex vivo methods for the prevention of complement activation byartificial, extracorporal surfaces (e.g. surface coatings), anunspecific complement activation was successfully carried out. Further,selective removal of activated complement factors making use of specificC5 antibodies is known from U.S. Pat. No. 5.853,722 and certainly alsoto be preferred, especially as highly affined antibodies have beengenerated in the meantime against all the components of the complementsystem.

The functional cascade manifested is primarily used to eliminate thebacteria penetrating into the organism. But as soon as a discrepancyoccurs between the number and/or virulence of the penetrating bacteriaand the elimination capacity of the immune system (e.g. inpost-traumatic immune deficiency), an excessive activation is observed,subsequently accompanied by a massive release of “shock mediators”(interleukins, thrombocyte activation factor (PAF), but also oxygenradicals, prostagiandins and their metabolic products), thus furtherlimiting the elimination capacity for LPS. In addition, CD14-negativecells (e.g. endothellae) are also activated by the LPS, as soluble CD14(sCD14) exists in the blood plasma as an LPS trapper, facilitatingbinding to these cells and inducing the formation and release of furthershock mediators, thus reinforcing the circulus vitiosus. As the shockmediators act selectively, but not specifically, function restrictionsin various cells and organs are observed (blood coagulation system,circulation, complement system), with the result that the inflammationreactions attacking the entire organisms initiate shock genesis, leadingto irreversible organ damage, to circulation collapse and death.

In order to break through this chain of functions, various therapystrategies have been studies.

Interruption of the cascade with antibodies interrupting the LPS bindingto proteins (LBP, sCD14), to the receptor (CD14), to released cytokinesor to cytokine receptors or with antagonists blocking the functionalareas of the receptors did achieve impressive success in various sepsismodels in animal experiments, but there are still no clinically tested,successful prevention and/or therapy studies.

It was not possible to fulfil the high expectations, as it wasincreasingly seen that LPS also influences and changes the functionalcondition of cells and tissue which are not impaired by thesetherapeutic approaches. In addition, it must be taken into account thatan LPS (immune complex) inactivated by an antibody/antagonist must beeliminated in order to exclude a biological reactivity on a permanentbasis. But the elimination is also a function of the immune system,which, as it is greatly weakened, can hardly or only very imcompletelyfulfil this task.

The development of the septic shock is a very dynamic occurrence ofprimarily varying genesis, in which various mediators cause highlydiffering reactions within a short period of time, these quickly leadingto the expression of the septic shock by dysregulation after an initiallife-maintaining function.

Therefore, the invention was based on the task of developing animmunoadsorption system of modular construction, in particular forextra-corporal detoxification, enabling a reduction of the plasma andtissue levels specific to the patient.

Inter alia, the invention is based on the knowledge that TNFα has a keyrole to play in this regulation system. It is released inter alia bymacrophages as a result of various “external” influences such asinjuries, inflammations, infections, septicaemia and induces a local andsystemic activation of the unspecific and specific defence system via acytokine cascade (IL-1, IL-6). Clinically, a massive TNFα release isexpressed by increased body temperature, lack of appetite and all thesubsequent symptoms of a catabolic metabolism situation. In pathogenesisof the sepsis, activation of the macrophages and thus the release ofTNFα- appears to be of essential importance for a survival of thepatient in the early phase of this disease, whereas the continued stateof activation results in the de-compensation of all defence reactions inthe further course.

The task of the invention was solved by an immunoadsorber for use insepsis therapy. The immunoadsorber according to the invention isparticularly used for the removal of complement factors andlipopolysaccharides (LPS) as well as the removal of further sepsismediators, and also TNF and interleukins from body fluids, if need be.It is characterised by carrier materials of organic or syntheticpolymers, to which both poly or monoclonal antibodies aimed against thecomplement factors C3a and/or C5a, and also antibodies aimed againstlipopolysaccharides (LPS) are bound. In a preferred embodiment,antibodies aimed against further sepsis mediators are also bound to thecarrier.

Preferably, these are polyclonal antibodies, particularly preferablyavian antibodies of type IgY. The antibodies against sepsis mediatorsare contained according to the state of the dysregulation.

According to this invention, these are antibodies aimed against TNF,IL1, IL6, IL8 and/or IL 10.

Preferred antibodies against the complement factor C3a manifest specificactivity against at least one of the following peptide sequences:

-   NH₂-KCCEDGMRQNPMR-COOH (SEQ ID NO: 1)-   NH₂-RFSCQRRTRFISL-COOH (SEQ ID NO: 2)-   NH₂-ITELRRQHARAS-COOH (SEQ ID NO: 3)

Preferred antibodies against the complement factor C5a possess specificactivity against at least one of the following peptide sequences:

-   NH₂-QADYKDDDDKLPAE-COOH (SEQ ID NO: 4)-   NH₂-DDKLPAEGLDIENS-COOH (SEQ ID NO: 5)

Preferred antibodies against IL1 α/β possess specific activity againstat least one of the following peptide sequences:

-   NH₂-NCYSENEEDSSSID-COOH (SEQ ID NO: 6)-   NH2 GAYKSSKDDAKIT-COOH (SEQ ID NO: 7)-   NH₂-WETHGTKNYFTS-COOH (SEQ ID NO: 8)-   NH₂-RISDHHYSKGFRQA-COOH (SEQ ID NO: 9)-   NH₂-VQGEESNDKIPVA-COOH (SEQ ID NO: 10)-   NH₂-ESVDPKNYPKKKMEKRF-COOH (SEQ ID NO: 11)

Preferred antibodies against IL6 possess specific activity against atleast one of the following peptide sequences:

-   NH₂-APHRQPLTSSERIDKQI-COOH (SEQ ID NO: 12)-   NH₂-QNRFESSEEQARA-COOH (SEQ ID NO: 13)-   NH₂-AITTPDPTTNAS-COOH (SEQ ID NO: 14)

Preferred antibodies against IL10 possess specific activity against atleast one of the following peptide sequences:

-   NH₂-SPGQGTQSENSCT-COOH (SEQ ID NO: 15)-   NH₂-QMKDQLDNLLLKES-COOH (SEQ ID NO: 16)-   NH₂-MPQAENQDPDIKA-COOH (SEQ ID NO: 17)-   NH₂-LPCENKSKAVEQ-COOH (SEQ ID NO: 18)

Preferred antibodies against TNFα possess specific activity against atleast one of the following peptide sequences:

-   NH₂-VRSSSRTPSDKPVA-COOH (SEQ ID NO: 19)-   NH₂-KSPCQRETPEGAEAKPW-COOH (SEQ ID NO: 20)

The immunoadsorber according to the invention manifests membranes orparticles customary per se of organic or synthetic polymers as carriermaterials, e.g. of polystyrenes, carbohydrates such as cellulose oragarose derivatives, or of acrylates, with the specific antibodies beingcovalently linked to them or fixed to them via spacers or linkers.

The production of the immunoadsorbers according to the invention is doneby methods known per se in that the antibodies aimed against C3a and/orC5a and LPS and, if need be, against further sepsis mediators arecoupled covalently or adsorptively to the carrier materials or organicor synthetic polymers.

The specific antibodies are produced by immunisation known per se,preferably of small mammals such as mice, rats or rabbits, or birds,such as chickens, with the corresponding antigens.

The object of the invention is also the use of the immunoadsorbers inappliances for the removal of complement factors, LPS and, if need be,further mediators from body fluids such as blood plasma as a function ofthe patient-specific situation.

Preferably, the immunoadsorbers are used in sepsis therapy forpiasmapherese in patients with sepsis or septic shock.

Although antibodies are available for most substances and are coupled tothe various carriers by known methods, avian antibodies are preferablyused, as they do not activate the complement system, unlike mammalantibodies. As the activating properties are bound to the F_(c) part ofthe mammal antibodies, the F_(ab) fragment fissured with papain canprincipally also be used.

According to the current state of knowledge, immobilised avianantibodies have no kind of unspecific effects on the human defencesystem. Birds, preferably chickens, are immunised with customary methodswith or without the use of adjuvants. The specific immunoglobulins areexcreted in the egg yolk and can be isolated from it with customarymethods. They are covalently bound to micro-particles or membranes viathe Fc part with known methods.

With the immunoadsorption system for extra-corporal detoxificationaccording to the invention, there exists for the first time a selectivesystem which can be used patient-specifically and by whichdysregulations of the immune system can be rectified.

The invention is explained in more detail by the following examples:

EXAMPLE 1

Production of polyclonal antibodies by means of immunogenic peptides:

TABLE I The peptides listed in Table I are produced by means of a solidphase synthesis Peptide sequence Antigen KCCEDGMRQNPMR (SEQ ID NO: 1)C3a RFSCQRRTRFISL (SEQ ID NO: 2) ITELRRQHARAS (SEQ ID NO: 3)QADYKDDDDKLPAE (SEQ ID NO: 4) C5a DDKLPAEGLDIENS (SEQ ID NO: 5)SPGQGTQSENSCT (SEQ ID NO: 15) IL10 QMKDQLDNLLLKES (SEQ ID NO: 16)MPQAENQDPDIKA (SEQ ID NO: 17) LPCENKSKAVEQ (SEQ ID NO: 18)NCYSENEEDSSSID (SEQ ID NO: 6) IL1 α GAYKSSKDDAKIT (SEQ ID NO: 7)WETHGTKNYFTS (SEQ ID NO: 8) RISDHHYSKGFRQA (SEQ ID NO: 9) IL1 βVQGEESNDKIPVA (SEQ ID NO: 10) ESVDPKNYPKKKMEKRF (SEQ ID NO: 11)APHRQPLTSSERIDKQI (SEQ ID NO: 12) IL6 QNRFESSEEQARA (SEQ ID NO: 13)AITTPDPTTNAS (SEQ ID NO: 14) VRSSSRTPSDKPVA (SEQ ID NO: 19) TNFαKSPCQRETPEGAEAKPW (SEQ ID NO: 20) These peptides are covalently bound toa carrier (KLH) according to a standard recipe. The conjugate dissolvedin PBS is mixed in equal shares with Freund's adjuvant. The individualinoculation dose is set in such a way that it contains 200 pg of thepeptide belonging to the antigen in question. 15-week-old young hens areim-

EXAMPLE 2

Production of Polyclonal Antibodies by Means of Lipopolysaccharides(LPS)

Cleaned LPS (SIGMA) of E. coli J5 are dissolved in PBS and mixed inequal shares with Fueund's adjuvant. 15-week-old young hens areimmunised with this mixture. The LPS dose amounts to 1 mg of LPS perimmunisation. Boostering is done 4 times at intervals of 4 weeks.

EXAMPLE 3

Obtaining the Antibodies (IgY) from Egg-Yolk:

The eggs from the clutches of the immunised hens are collected. Afterseparation of the egg-yolk containing antibodies, there is storage at−20° C. According to requirements, the yolks are thawed and treatedaccording to the following plan (C. SCHWARZKOPF, B. THIELE (1996) ALTEX13 Suppl. 16, 35-3):

-   -   A TBS: 20 mM Tris/HCl, pH 7.5, 0.5 M NaCl    -   B 10% (w/v) dextra sulphate in A Solutions    -   C 1 M CaCl₂    -   D 0.5 M EDTA, pH 7.5    -   E saturated ammonium sulphate solution

The egg yolk (corresponds to a volume of 10-20 ml/egg-yolk) is suspendedin 100 ml TBS per egg-yolk. Lipids and lipoproteins are precipitatedwith dextran sulphate (6 ml B per 100 ml TBS/egg yolk suspension) andCa⁺⁺ (15 ml C per 100 ml TBS-egg yolk suspension), stirred for 30 to 60min. at room temperature and centrifuged off at 5,000 g. The pellet iswashed with a small volume of TBS (approx. 20 mlg/egg yolk) andcentrifuged again.

The combined supernatants are filtered through a paper filter, then 0.5M EDTA is added to the filtrate up to a final concentration of approx.30 mM EDTA (6 ml per 100 ml), in order to bind remaining Ca⁺⁺ ions.After this, the supernatant is mixed with 24.3 g of ammonium sulphateper 100 ml (corresponds to 40% saturation) and incubated at +4° C. for30 min.. The resultant precipitation (IgY) is firstly washed with 30%(NH₄)₂SO₄ (30 ml E+70 ml dist. water), centrifuged, then dissolved inthe smallest possible volume of TBS (approx. 10 ml/egg-yolk used) anddialysed against TBS.

The content of IgY is determined photometrically at 275 nm.

EXAMPLE 4

a) Activation of a Carrier:

The IgY cleaned according to Example 3 are covalently bound to asuitable carrier. For example, sepharose can be activated as describedbelow for this purpose (H.- F. Boeden, W. Büttner, C. Rupprich, B.Büttner, S. Heinrich, M. Becker, M. Holtzhauer (1992) Makromol. Chem.193, 865-887):

The agarose carrier is gradually transformed, i.e. with an amount ofacetone increasing in steps of 20%. Finally, the carrier is left tostand in an enclosed container in a quintuple bed volume with water-freeacetone overnight, again washed with 5 to 10 Vol. water-free acetone andbriefly sucked off on a G2 slice. 400 mgN-(Chlorcarbonyloxy)-5-norbornen-2,3-dicarboximid (CICOONB) in 10 mlwater-free acetone p.a. are added to 10 ml sedimented carrier. Within 15minutes, a solution of 280 μl triethylamine and 20 mg4-dimethylamino-pyridine (DMAP) in 5 ml dry acetone is added drop bydrop (mol ratio CICOONB:triethylamine:DMAP 1:1.2:0.1) with shaking.After this, there is further shaking for 15 minutes, after which thecarrier is washed with about 200 ml water-free acetone.

b) Coupling of the IgY to a Solid Carrier:

The polysaccharide matrix (gel) activated according to Example 4a) isgradually transformed into a watery medium and then immediately stirredinto the coupling solution containing the ligand. Citrate buffer pH 4.2is used as a coupling buffer. The coupling is done with gentle shakingfor 2 h at room temperature. Free bindings are subsequently blocked byaddition of ethanolamine. Table 2 shows the concrete conditions for theindividual antibodies.

TABLE 2 Ab ml coupling Ethanol- solu- buffer amine moist Gel Chicken-mg/ tion (Citrate, 0.1 1 M gel No. Ab (lgY) mg ml (ml) M, pH 4.2) (ml)(g) 1 ChaIL1 9.5 13.5 0.7 4.3 0.5 5.55 2 ChaIL6 9.8 9.8 1.0 4.0 0.5 5.583 ChaIL10 9.2 7.4 1.2 3.8 0.5 5.55 4 ChaTNF 11.0 11.6 1.0 4.1 0.5 5.56 5ChaLPS 11.6 13.7 0.9 4.2 0.5 5.60 6 ChaC3a 6.9 10.7 0.6 4.4 0.5 5.57 7ChaC5a 11.3 11.1 1.0 4.0 0.5 5.55 8 Control 0.0 0.0 0.0 5.0 0.5 5.61

EXAMPLE 5

The antibodies immobilised according to Example 4 are used in order toremove lipopolysaccharides, interleukins, TNF or complement factors fromliquid media such as buffer solutions, serum or blood plasma.

For this, the carriers are washed, transformed into a physiologicalbuffer (PBS) and packed in plastic or glass pillars free of air bubbles.The arrangement is completed by connection to a chromatographyappliance. The sample material to be adsorbed (buffer doted with theantigens, serum or blood plasma samples, doted or with natural antigencontent) can now be guided by gravity or with a suitable pump via theimmobilised antibodies specific for the antigens stated. The existingantigens are recognised, firmly bound and thus removed from the mediumflowing through the column by the IgY. The detection of the effectivityis done by analysis (ELISA) of the column throughflow, the antigencontent of which is reduced. After washing of the column with aphysiological buffer, there is desorption of the bound antigen withsuitable elution agents (0.1 M citrate buffer pH 3), fractioning andanalysis of the eluate. Quantitative detection of the antigens is usedto determine the capacity of the immunosorbent.

1. An immunoadsorber for blood treatment use in sepsis therapy, theimmunoadsorber comprising a carrier of organic or synthetic polymers towhich are immobilized antibodies that are specific to C3a and/or C5a andto lipopolysaccharides (LPS) and wherein, a) the antibodies to C3a arespecific for at least one peptide selected from the group consisting ofSEQ ID NO: 1, 2, and 3; and b) the antibodies to C5a are specific for atleast one peptide selected from the group consisting of SEQ ID NO: 4,and
 5. 2. The immunoadsorber according to claim 1, wherein theantibodies are polyclonal antibodies.
 3. The immunoadsorber according toclaim 2, wherein the antibodies are avian antibodies of type IgY.
 4. Theimmunoadsorber according to claim 1, further comprising at least oneimmobilized antibody specific for at least one sepsis mediator selectedfrom the group consisting of TNF, 1L1, 1L6 , IL8 and/or IL10.
 5. Theimmunoadsorber of claim 4, wherein the immobilized antibodies comprisingthe immunoadsorber are varied as a function of the actual content ofsepsis mediators in the blood.
 6. The immunoadsorber according to claim4, wherein the immobilized antibodies are specific for at least one ofthe following peptide sequences of interleukins 1α and 1β IL1α:NH2-NCYSENEEDSSSID-COOH SEQ ID NO. 6 NH2-GAYKSSKDDAKIT-COOH SEQ ID NO. 7NH2-WETHGTKNYFTS-COOH SEQ ID NO. 8 ILβ: NH2-RISDHHYSKGFRQA-COOH SEQ IDNO. 9 NH2-VQGEESNDKIPVA-COOH SEQ ID NO. 10 NH2-ESVDPKNYPKKKMEKRF-COOHSEQ ID NO.
 11. 7. The immunoadsorber according to claim 4, wherein theimmobilized antibodies are specific for at least one of the followingpeptide sequences of interleukin 6: IL6: NH2-APHRQPLTSSERIDKQI-COOH SEQID NO. 12 NH₂-QNRFESSEEQARA-COOH SEQ ID NO. 13 NH2-AITTPDPTTNAS-COOH SEQID NO.
 14. 8. The immunoadsorber according to claim 4, wherein theimmobilized antibodies are specific for at least one of the followingpeptide sequences of interleukin 10 IL10: NH2-SPGQGTQSENSCT-COOH SEQ IDNO. 15 NH2-QMKDQLDNLLLKES-COOH SEQ ID NO. 16 NH2-MPQAENQDPDIKA-COOH SEQID NO. 17 NH2-LPCENKSKAVEQ-COOH SEQ ID NO.
 18. 9. The immunoadsorberaccording to claim 4, wherein the immobilized antibodies are specificfor at least one of the following peptide sequences of TNFα TNFα:NH2-VRSSSRTPSDKPVA-COOH SEQ ID NO. 19 NH2-KSPCQRETPEGAEAKPW-COOH SEQ IDNO.
 20. 10. The immunoadsorber according to claim 1, wherein the organicor synthetic polymers further comprise membranes or particles of one ormore of the group consisting of polystyrenes, carbohydrates, cellulose,agarose derivatives, and acrylates.
 11. The immunoadsorber according toclaim 1, wherein the immobilized antibodies are covalently bound to thecarrier.
 12. The immunoadsorber according to claim 1, wherein theimmobilized antibodies are attached to the carrier via spacers orlinkers.
 13. A method for the production of immunoadsorber according toclaim 1, wherein antibodies specific for C3a and/or C5a and LPS and,optionally, against further sepsis mediators are covalently oradsorptively coupled to the carrier.
 14. A method according to claim 13,wherein the antibodies are produced by immunisation of mammals or birdswith the corresponding antigens.
 15. The method of claim 14 wherein theantibodies are raised by immunizing one or more animals selected fromthe group consisting of mice, rats, rabbits or chickens.
 16. A method oftreating blood plasma or serum using the immunoadsorber of claim 1, themethod comprising the steps of, providing an amount of blood plasma orserum in need of sepsis therapy; and contacting the blood with theimmunoadsorber of claim 1; and recovering the contacted blood plasma orserum from the immunoadsorber.
 17. The method of claim 16, wherein theblood plasma or serum has not been subjected to hemofiltration prior tocontacting the immunoadsorber.